Genetic targeting of cortical pyramidal neuron subtypes in the mouse Abstract A key obstacle to studying the development, organization, and function of neural circuits in the cerebral cortex is the stunning diversity of neuron types and a lack of comprehensive knowledge about their basic biology. The problem of neuronal diversity and identity in the cortex is fundamental, transcending developmental and systems neuroscience, and lies at the heart of defining the biology of cognition and psychiatric disorders. Glutamatergic pyramidal neurons (PyNs) constitute ~80% of cortical neurons, are endowed with large capacity for information coding, storage, plasticity, and carry the output of cortical computation. PyNs consist of diverse subtypes based on their specific laminar locations, axonal projection patterns, and gene expression profiles. Subsets of PyNs form multiple and hierarchical subnetworks of information processing, with distinct output channels to cortical and subcortical targets that subserve sensory, motor, cognitive and emotional functions. Importantly, PyN subtypes are differentially affected in various neuropsychiatric and neurodegenerative disorders. However, the severe lack of specific and effective genetic tools for studying PyNs has hampered progress in understanding cortical circuits. We propose to build a comprehensive genetic tool set for major PyN subtypes in the mouse through a joint project led by Dr. Josh Huang at Cold Spring Harbor Laboratory and Dr. Paola Arlotta at Harvard University with key collaboration from Dr. Hongkui Zeng at the Allen Institute for Brain Science. We have discovered a set of specific and combinatorial markers that distinguish major PyNs subtypes. We will use intersection, subtraction, and inducible strategies to target PyN subtypes. Aim 1 will generate ~25 knockin Cre and Flp driver lines that target major subclasses and lineages of pyramidal neurons. Aim 2 will generate multiple intersectional and subtractive reporters that are broadly useful for labeling distinct neuronal subtypes. Aim 3 will characterize the specificity of drivers, and organize and display data and resource in public databases. We will combine anatomical tracing with mRNA in situ and immunocytochemistry to determine the specificity of genetic targeting. We will use high- throughput and high- resolution pipeline at the Allen Institut to characterize PyN subtype labeling and axon projections. We will deposit all tools and reagents in major repositories (JAX) that are publically accessible (Allen Brain Atlas website). Genetic targeting will provide entry points to cortical circuits by integrating the full range of modern technologies and facilitate a systematic and comprehensive analysis of PyNs, from cell fate specification to circuit integration, connectivity, and function in cortical processing and behavior. These genetic tools will further provide sensitive probes to pathogenic mechanisms in models of brain disorders including autism, schizophrenia, bipolar and ALS, epilepsy, Alzheimer's, dementia, and may yield cell and molecular targets for therapeutic intervention. Although this grant is not hypothesis-driven as a traditional R01, it is certainly hypothesis-enabling for many future R01s to elucidate the fundamental role of the cerebral cortex in the larger framework of CNS architecture and function. 1